Dietary fibers are important for their hypoglycemic effect, hypolipidemic effect; lowering serum cholesterol
hence helps in prevention of atherosclerosis, antitoxic effect and anti-cancerous effect. It also helps in control of
gastro intestinal disorders like gall stone, irritable bowel syndrome, constipation, inflammatory bowel disease etc.
Beneficial effects of cereal fibers are frequently discussed in the context of whole grain consumption; unrefined
whole grains and bran products are highly complex substances containing both soluble and insoluble dietary fiber as
well as other biologically active substances e.g. polyphenols, antioxidants, vitamins, trace minerals, phytoestrogens,
lipids, proteins, and starch. Research on minor millets and its food value is in its infancy and its potential vastly
untapped. So the present study was undertaken to evaluate the in-vitro hypoglycemic effect of insoluble fibers
from locally available whole grain of millets and cereals like kodo millet (Paspalum scrobiculatum), Proso millet
(Panicum miliaceum), Barnyard millet (Echinochloa frumentaceae), Finger millet (Elusine coracana), wheat (Triticum
aestivum) and Great millet (Sorghum vulgare) from tribal belt of Odisha. Proximate analysis of the cereals and
millet grains revealed that these grains are rich in crude fiber, total ash and crude protein content. The nutritional
composition is better than most of the commonly used grains. In general the crude fiber and ash content of the bran
samples were more as compared to the grains. Glucose adsorption capacity (GAC) at 5 Millimole/l concentration
of glucose was almost similar in IDF of all the millets and wheat ranging from 0.04 ± 0.01 in case of Barnyard millet
IDF to 0.06 ± 0.01 in Sorghum, Ragi and Kodo. Glucose absorption capacity at 5 Millimole/l concentration of glucose
was highest in Ragi fibers but at higher concentration of glucose it was highest in wheat fibres. GAC increases with
increase in glucose concentration in all the cases studied. In most samples WIS showed maximum GAC. Maximum
GAC was found at 50 mM/lit in Ragi (Finger millet) IDF and lowest value was found at 10 mM/lit in jhipiri (Barnyard
millet) IDF and wheat AIS. In case of glucose diffusion and GDRI, in all types of fiber, it showed decrease in glucose
concentration in the dialysate with addition of fiber than in control (without fiber), indicating that addition of fiber
decreased diffusion of glucose through dialysis membrane which simulates the function of membrane of small
intestine. The glucose concentration in the dialysate though increases with increase in time but remains lower to that
of glucose value in control. When GDRI values were compared it showed lowest value in case of IDF than AIS and
WIS in all the six samples. Effect of insoluble fibers on alpha-amylase activity indicate that glucose production rate
is highest in kodo (kodo millet) AIS but lowest in sorghum (Great millet) IDF. When residual amylase activity was
compared it showed highest values in Ragi (Finger millet) AIS and lowest values in gunji (Proso millet) IDF.

The present global pandemic of Diabetes is accounted for by
westernization of life style, population growth, ageing and urbanization,
with consequent dietary change, sedentary life style and obesity.
Diabetes Mellitus(DM) is also a common endocrine disease in middle
aged to older cats and is often called as “sugar diabetes’’. In dogs with
naturally occurring insulin dependent DM a high insoluble fiber diet
may aid in glycemic control. Incidence of DM is lower in population
with high fiber intake mostly in rural areas and tribal belt which might
be due to feeding of different cereals and millets with high fiber content.
The term dietary fiber was first adopted in 1953 by Hipsley to describe
the plant cell wall components of food. Millets are small sized grains,
containing large proportions of husk and bran; require dehusking and
debranning prior to consumption [1]. The nutritive value of millets is
comparable to other cereals, some of them are even better with regard
to average protein and mineral contents [2].

The protein contents of the dehusked millets varied between 8.7%
(kodo millet) and 13.8% (pearl millet), whereas in case of milled grains
it varied from 5.8% (finger millet) to 12.7% (pearl millet). Milled grains
contained nearly 70% of total fat of whole seeds. The calcium and
phosphorus contents of milled millets varied from 2.3 mg% to 162.8 mg% and 105 mg% to 425 mg%, respectively. Milling removed nearly
50% calcium and about 65% of phosphorus from whole seeds. The
bran fraction from small millets other than finger millet, contain 23.0-
27.0% oil. Whereas pearl millet bran contain 15% oil. The total dietary
fiber content of debranned millets is ranging from 9.0 to 16.0 %. This
indicates the millets, even after removal of husk and major portion of
bran, contained appreciable amounts of dietary fiber. The millet bran,
besides containing considerably higher proportion of oil, appears to be
a good source of dietary fiber, out of which 10-15% was soluble fraction
[3].

Dietary fibers are not uniform chemically or in their nutritive and
biological properties, the only common ground being their resistance to mammalian digestive enzymes. The AOAC [4] method for total fiber
is subjected to interference from ash, protein, tannins, and resistant
starches. These interferences can be reduced by urea enzymatic dialysis.
The measurement of soluble and insoluble fiber is nutritionally relevant
since physical properties greatly modify dietary effects of fiber.
Insoluble fiber is conveniently measured as neutral detergent fiber. This
procedure has been improved by reducing the starch interference and
the time of analysis. Physical and biological properties of dietary fiber
can be measured by using relevant procedures for hydration capacity
and rate of fermentation. The lignin and tannin content modify the
characteristics of dietary fiber [5]. Breads made from a combination of
wheat potato and/or oat has relatively high in total dietary fiber. Wheat
breads with different ash contents or breads made from a combination
of wheat and rye had clearly higher total dietary fiber content [6].
Schieber et al. [7] has pointed out that agricultural byproducts could be
exploited as a potential source of fibers and functional compounds for
food application. Dietary fiber is unique among feed constituents
because it is defined only on a nutritional basis (i.e. in terms of digestive
and physiological effects that it elicits) but must be measured
chemically. The usefulness of dietary fiber results vary from its value as
an indicator of physiological health benefits to its value as a predictor of
digestibility and energy value of feeds. Numerous methods have been
proposed for measuring dietary fiber and some have become routine
analyses for research and practical use. Fiber extraction methods are
typically categorized in to three types (chemical-gravimetric,
enzymatic-gravimetric or enzymatic-chemical) based on ways fibrous
residues are isolated and measured. Isolation of dietary fiber residue is
done by extraction in chemical solution, enzymatic hydrolysis of nonfibrous
constituents or a combination of two. After fibrous residue is
isolated it is measured either gravimetrically (weighing the residue) or
chemically (hydrolyzing the residue and measuring individual
components such as sugars and lignin) [8]. There are several AOAC
official methods for measuring total dietary fiber (TDF), Insoluble
dietary fiber (IDF), soluble dietary fiber (SDF). The first AOAC method
for TDF is 985.29–Total dietary fiber in food, enzymatic gravimetric
method which did not allow separation of dietary fiber in to soluble
and insoluble fraction. Insoluble fraction can be determined using
AOAC official method 991.42–Insoluble dietary fiber in foods and food
products, enzymatic gravimetric method [8]. Certain physiological
responses have been associated with the consumption of dietary fiber
and physical and chemical properties of individual dietary fiber
compounds appear to be important in determining the physiological
response to sources of dietary fiber in the diet [9]. The post prandial
glycemic response is most effectively reduced with sources of viscous
polysaccharides. The importance of viscosity in this response has been
demonstrated in several studies suggesting that the ability to form a gel
matrix may be important in mediating the physiological response to
these fiber sources. Also the physical properties of digestibility are key
determinants in the metabolism of fiber rich diets. Fiber modulates and
slows the rate of digestion and absorption by at least three mechanisms.
1) The rate of gastric filling and emptying can be slowed by certain
fibers; 2) The activity of digestive enzymes in the small intestine could
be diminished in presence of fibers; 3) The diffusion and absorption of
nutrients enzymes and substrates in the intestinal tract may be altered
by certain fibers via these mechanisms and its fermentation in the large
intestine. Fibers affect the metabolic process [10]. The fermentability of
polysaccharides as well as their bulking ability in the large bowel is
important for determining the physiological effects of soluble versus
insoluble fibers. In this part of gut soluble fibers can be readily degraded
by bacteria because the water holding capacity allows the bacteria
penetrate the fiber matrix. The increase in bacterial mass due to fermentability leads to increase in faecal bulk. In contrast insoluble
fiber cannot be penetrated as well by bacteria and cannot be broken
down as extensively hence a residual fiber is present and a matrix is
maintained in large bowel content and bacterial mass is increased [11].
The rate at which food is emptied from the stomach determines the rate
of nutrient absorption from the intestine. Hence a delay in gastric
emptying determines the rate of nutrient absorption from the intestine
[9]. The findings that several dietary fibers can decrease the activity of
human pancreatic amylase, lipase, trypsin, and chymotrypsin may be
attributable at least in part to enzyme inhibitors [12]. Alternatively this
decreased activity after incubation with several dietary fibers could be
due to nonspecific adsorption of enzyme molecules [13]. With high
fiber intake glucose absorption is slowed down and spread out along a
greater length of the intestine. This allows uptake of glucose by the
intestine keeping in pace with the gastrointestinal absorption after
initial stimulation of insulin release there by regulating plasma glucose
level [14-16]. Dietary fibers coming from various sources do not seem
to be equally effective in delaying glucose absorption in intestine.
Tanchoco et al. [17] It is estimated that more than 50% post prandial
insulin secretion is triggered by intestinal peptide hormone. In presence
of elevated blood glucose, glucagons like peptide 1 (GLP-1) stimulates
the release of insulin by interacting with specific receptors on pancreatic
beta cells. In addition to potentiating glucose induced insulin secretion
GLP-1 stimulates proinsulin gene expression and proinsulin
biosynthesis [18]. By stimulating insulin release and increasing insulin
dependent glucose disposal, GLP-1 enhances glucose tolerance [19].
The potential action of this hormone on carbohydrate metabolism
makes it potentially applicable in the treatment of non-insulin
dependent diabetes mellitus. It is found that long term ingestion of
dietary fiber by rats ingesting similar amounts of energy, proteins,
lipid, glucose, vitamins and minerals stimulates small chain fatty acids
(SCFA) production and proglucagon m-RNA abundance and increases
post prandial glucagons like peptide 1, insulin and c-peptide
concentration. High carbohydrate/high fiber diet significantly
improves blood glucose control and reduces plasma cholesterol levels
in diabetic patients compared with a low carbohydrate low fiber diet. In
addition a high carbohydrate/high fiber diet does not increase plasma
and triglyceride concentration despite the higher consumption of
carbohydrate. Ability of dietary fiber to retard food digestion and
nutrient absorption certainly has an important influence on lipid and
carbohydrate metabolism. The fiber content and physical form of the
food can influence the accessibility of nutrients by digestive enzymes
thus delaying digestion and absorption. The fiber content and physical
form of the food can influence the accessibility of nutrients by digestive
enzymes thus delaying digestion and absorption. The identification of
these foods with a low glycemic response would help to enlarge the list
of foods particularly suitable for diabetic patients [20]. The
polysaccharides composing the major part of dietary fiber in fruits and
vegetables are beneficial to diabetes and heart patients since the fibers
lower blood sugar and serum cholesterol levels [21,22]. In fact the most
likely explanation for the reduction of post prandial hyperglycemia by
viscous fibers is decreased amylase activity [23] and a direct delaying
effect on glucose absorption in the gastro-intestinal tract due to
alternation in the diffusion of digestion end product with the lumen
[24,25]. Starch degradation and glucose diffusion are delayed in the
presence of mango fiber. Viscous solutions which reduce starch
digestibility in vitro can decrease glycemic post prandial response.
Hence mango fiber could be of potential benefit in controlling plasma
glucose [26].

Materials and Methods

Collection of sample

Grain samples of different millets and cereals (Table1) were
collected from Boudh district, Odisha. These samples were then cleaned
properly, shed dried and grinded manually and sieved to collect the
bran. Whole grain was grinded using electrically operated grinder to
desired size. Then these bran samples and whole grain powder samples
were stored in sealed containers till their use in different experimental
procedures (Figure 1,2).

Estimation of moisture content: Dry weight of moisture cup (W1)
was first taken. Then weight of moisture cup with bran/whole grain
sample (W2) was taken and it was kept in hot air oven over night (12
hrs) at 100°C. Weight of moisture cup with sample was again taken
after drying (W3). Moisture content is calculated as

Estimation of ether extract: The instrument used for estimation
of ether extract was Socs plus (Pelican Equipments, Chennai, India).
Moisture free samples (2 gm each) from the above experiment were
taken in different thimbles. Dry weight of flasks was taken (W1). The
thimbles were placed in the flasks. 150 ml of petroleum ether (60-80°C)
was taken in each flask. The attachment of the instrument were made
properly and was run for 1 hr at 90°C and further for 30 minutes at
180°C.

After collection of ether extractives the flask were removed from
the apparatus and kept in the hot air oven (100°C) for 12hrs to make
the flasks free from petroleum ether. The weight of flasks was taken
again (W2) and ether extract percentage was calculated by using the
formula

Estimation of crude fiber (by Von Soest method): Fat free samples
(W1, 2 gm each) were taken in the sintered crucible of Fibra Plus Fes 6
Instrument (Pelican equipment’s, Chennai, India). Then it was treated
with 1.25% H2SO4 (v/v) at 400°C for 45 min for acid digestion followed
by alkali digestion with 1.25% NaOH (W/V) at 400°C for 45 minutes.
Then the crucible containing fiber sample was washed with distilled
water and dried in oven at 100°C for 24 hrs. Then weighed (W2) and
placed in muffle furnace at 550°C for 6 hr and weighed (W3) again.
Percentage of crude fiber was determined by formula

Estimation of crude protein: Protein content of sample was
estimated using Nitrogen Autoanalyser manufactured by Pelican
Equipment’s, Chennai, India. 0.2 gms of dried sample, 3 gms kelpac, 10 ml H2SO4 were mixed and digested at 400°C for 3hrs using KES
06L Digestion Chamber of Pelican instrument. Distillation was done
by Kel Plus (Classic DX) apparatus manufactured by Pelican. Solutions
used in equipment were 40% NaOH and 4% Boric acid with distillation
time 9 minutes. Then the distillate was titrated against 0.1 N HCl. The
nitrogen content was titrated/estimated using Metrohm Autotitrator
(Switzerland). Protein percentage was calculated by multiplying
nitrogen content with 6.25.

Estimation of total ash: The dry weight of porcelain crucible
was taken (W1). Then weight of crucible with moisture free sample
was taken (W2) and kept in muffle furnace at 550°C for 6 hrs. After
cooling weight of crucible with ash content was taken (W3). Total ash%
calculated by formula

All the above procedures were followed both for bran and whole
grain powder of different millets and cereals to estimate their proximate
composition.

Preparation of insoluble dietary fiber (IDF) from bran sample

The moisture free bran samples were first made fat free by using
Socs Plus (Pelican Equipment, Chennai). The fat free samples were
used for extraction of IDF by Von Soest method with acid and alkali
treatment using Fibra plus Fes-6 (Pelican Equipment, Chennai). Acid
digestion of samples was performed at 400°C for 45 min using 1.25%
H2SO4 followed by Alkali digestion using 1.25% NaOH at 400°C for
45 min. Then the contents were filtered and washed thoroughly with
distilled water to make the sample free from alkali and kept in hot air
oven at 100°C for drying. Then the fiber samples were weighed and
stored till further use.

Preparation of alcohol insoluble solids (AIS) from bran
samples

Alcohol insoluble solids (AIS) were prepared from the bran sample
by using the method described by [27]. Briefly, 3 gms of bran sample
was homogenized with boiling alcohol (ethanol) 850 ml/lit at high
speed followed by further boiling for 40 min. The bran to alcohol ratio
was 1:30 (w/v). AIS was collected by filtration and washed with ethanol
(700 ml/lit), air dried and stored properly for further use.

Preparation of water insoluble solid (WIS) from bran sample

The water insoluble solids (WIS) were prepared according to the
method of [28]. WIS was separated from bran samples by homogenizing
the bran in cold distilled water (1:10 w/v) at high speed for 1 min. After
filtration WIS was washed with ethanol (700 ml/lit), air dried and
stored for further use.

Study of hypoglycemic activity in vitro

Determination of glucose adsorption capacity (GAC): The
glucose adsorption capacity (milimoles per gram) was determined
according to method described by [28] with slight modification. 0.1 gm
of fiber sample was mixed with 10 ml of glucose solution of different
concentration (i.e. 5 mmol/lit, 10 mmol/lit, 25 mmol/lit, and 50 mmol/
lit ) and then incubated for 5 hrs at 37°C using constant shaker cum
incubator (Rotek-LIS, Pelican Equipments, Chennai ). The final
glucose content in the supernatant was measured after centrifuging
at 3500 rpm for 15 min by glucose assay kit (Corals glucose assay kit,
GOD-POD method) to estimate the amount of glucose adsorbed on
fiber sample. A control test was done without addition of fiber.

Determination of glucose diffusion and glucose dialysis
retardation index (GDRI): Glucose dialysis retardation index was
determined on the basis of [28] with slight modifications. A mixture
solution was prepared by mixing 0.125 gm of fiber sample in 6.25 ml
of glucose solution (10 mmol/lit) and was dialyzed against 40 ml of
distilled water at 37°C using a dialysis membrane with a molecular
weight cut off value of 12,000 D. After incubation of 10, 30, 60, and
120 min the glucose content in the dialysate was measured by glucose
assay kit (Corals glucose assay kit, GOD-POD Method) for estimation
of GDRI. A control test was also prepared without addition of fiber.
GDRI was calculated using formula

Determination of starch digestibility: The effect of different
fibers on starch digestibility was determined as per [28] with slight
modifications. A mixture was prepared by mixing 0.1 gm of fiber
and 0.02 gm of diastase in 5 ml of potato starch solution (4 gm/100
ml) and was dialyzed against 100 ml of distilled water at 37°C using
a dialysis membrane with molecular weight cut off value of 12,000 D.
After incubation of 10, 30, 60 and 120 min the glucose content in the
dialysate was determined using glucose assay kit (Coral glucose assay
kit, GOD-POD Method). A control experiment was also performed
without addition of fiber.

Determination of residual amylase activity: The effect of fiber on
glucose production rate and residual amylase activity was determined as
per [28], with slight modifications. An incubation mixture containing
0.25 gm of fiber sample and 1 mg of diastase in 10 ml of potato starch
solution (4 gm/100 ml) was incubated at 37°C for 60 min. Starch
digestion then stopped by addition of 20 ml of 0.1 N NaOH. Then it was
centrifuged at 3500 rpm for 15 min and glucose content of supernatant
was measured by Glucose Assay kit (Corals glucose assay kit, GODPOD
method). A control experiment was also done without addition
of fiber. The residual amylase activity was defined as the percentage of
glucose production rate with fiber addition over the control.

Results

Proximate composition

The proximate composition of whole grains and bran samples were shown in percentage (Table 2 and 3) for whole grain and bran samples
respectively. Moisture content in whole grains varied slightly ranging
from 7.63 ± 0.581% to 8.89 ± 0.12%, barnyard millet being the highest in
moisture content and kodo millet being the lowest. Highest crude fiber
content (17.42 ± 1.066%) was found in finger millet and was lowest in
sorghum (10.48 ± 0.494%). The dry matter content was highest in kodo
millet (92.37 ± 0.581%) and lowest in barnyard millet (91.11 ± 0.068%).
The ether extractives were highest in barnyard millet (4.35 ± 0.164%)
and lowest in Proso millet (1.003 ± 0.057%) whereas, the crude protein
content was highest in barnyard millet (10.39 ± 0.248%) and was lowest
in kodo millet (5.48 ± 0.449%). The total ash content was found to be
highest in both kodo millet (3.59 ± 0.246%) and Proso millet (3.59 ±
0.234%) and lowest in sorghum (1.29 ± 0.085%). The NFE content was
highest in Sorghum (78.47 ± 0.968%) but lowest in barnyard millet
(70.47 ± 0.747%).

From the proximate composition of bran samples (Table 3) it was
observed that the moisture content was highest in finger millet (9.22
± 0.56%) and lowest in kodo millet (5.53 ± 0.67%) and the dry matter
content was highest in kodo millet (94.47 ± 0.67%) and lowest in Ragi
(90.78 ± 0.56%). The ether extract content was highest in sorghum
(4.69 ± 0.60%) but lowest in Proso millet (2.8 ± 0.53%). The crude
fiber content in bran samples varied from 38.4 ± 0.66% to 11.32 ±
0.73, barnyard millet bran being the highest and wheat bran being the
lowest whereas, the crude protein content was highest in wheat bran
(12.19 ± 0.62%) and was lowest in Proso millet bran (4.13 ± 0.56%). The
total ash content was found to be highest in finger millet bran (10.49 ±
0.85%) and lowest in kodo millet bran (7.74 ± 0.89%). The bran sample
of Sorghum was highest in NFE (63.51 ± 0.48%) but the barnyard millet
contained lowest NFE (43.29 ± 1.48%). From proximate composition
analysis of both whole grains and bran samples it was clear that the
crude fiber content was more in bran samples than the corresponding
whole grains so the bran samples were used for the extraction of fibers
for further studies.

Effect of insoluble fibers on glucose adsorption capacity in
vitro: A Series of different concentration of glucose (5 Millimole/l, 10
Millimole/l, 25 Millimole/l and 50 Millimole/l) were used to investigate
the in vitro glucose adsorption capacity (Table 4, Figure 3). The results
showed that all the dietary fibers could bind glucose and the adsorption
capacity also increases in almost all the samples studied with increase
in concentration of glucose. Glucose adsorption capacity (GAC) at 5
Millimole/l concentration of glucose was almost similar in IDF of all
the millets and wheat ranging from 0.04 ± 0.01 in case of Barnyard
millet IDF to 0.06 ± 0.01 in Sorghum, Ragi and Kodo. In case of
alcohol insoluble solids (AIS) glucose adsorption capacity (GAC)
at 5 Millimole/l concentration of glucose ranged from 0.04 ± 0.01 in
wheat to 0.10 ± 0.01 in Ragi and sorghum whereas, that in case of WIS
ranged from 0.07 ± 0.02 in Barnyard millet and wheat to 0.18 ± 0.01
in Ragi. Glucose absorption capacity at 5 Millimole/l concentration of
glucose was highest in Ragi fibers. Glucose adsorption capacity (GAC)
at 10 Millimole/l concentration of glucose was ranging from 0.12 ±
0.01 in case of Ragi IDF to 0.25 ± 0.02 in wheat which is interestingly
reverse as compared to the 5 Millimole/l concentration of glucose.
In case of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC) at 10 Millimole/l concentration of glucose ranged from 0.12
± 0.01 in Proso millet to 0.31 ± 0.02 in Ragi whereas, that in case of
WIS ranged from 0.08 ± 0.02 in Proso millet to 0.25 ± 0.02 in Ragi.
Glucose absorption capacity at 10 Millimole/l concentration of glucose
was interestingly highest in wheat fibers. Glucose adsorption capacity
(GAC) at 25 Millimole/l concentration of glucose was ranging from
0.19 ± 0.01 in case of kodo millet IDF to 0.71 ± 0.02 in wheat. In case
of alcohol insoluble solids (AIS) glucose adsorption capacity (GAC)
at 25 Millimole/l concentration of glucose ranged from 0.15 ± 0.02 in
Barnyard millet and Ragi to 0.62 ± 0.02 in wheat whereas, that in case of
WIS ranged from 0.08 ± 0.02 in Barnyard millet to 0.51 ± 0.02 in wheat.
Glucose absorption capacity at 25 Millimole/l concentration of glucose
was also highest in wheat fibers.

Table 4: Glucose adsorption capacity of various insoluble fibers in different concentration of glucose.

Figure 3: Glucose adsorption capacity of various insoluble fibers in different concentration of glucose.

Glucose adsorption capacity (GAC) at 50 Millimole/l concentration
of glucose was ranging from 0.51 ± 0.01 in case of Barnyard millet IDF
to 1.65 ± 0.02 in wheat. In case of alcohol insoluble solids (AIS) glucose
adsorption capacity (GAC) at 50 Millimole/l concentration of glucose ranged from 0.3 ± 0.02 in Barnyard millet to 1.31 ± 0.01 in Sorghum
(Great millet) whereas, that in case of WIS ranged from 0.26 ± 0.01 in
Barnyard millet to 1.02 ± 0.05 in wheat. Glucose absorption capacity
at 50 Millimole/l concentration of glucose was also highest in wheat
fibers amongst all the fibres studied. From the results it was clear that
at 5 mM/lit, the GAC was more for WIS in all sample except in Kodo
millet, but in 50 mM/lit the GAC was highest for IDF in all six samples
studied.

Effect of insoluble fibers on glucose diffusion: (Table 5 and Figure
4) showed the variation in glucose diffusion with addition of insoluble
fibers compared to that of control as a function of time. With increases
in time from 10 to 120 min the glucose content in the dialysate with
addition of various fiber samples were increased in all six samples.
When compared with control, test of all the fibers from six different
samples could significantly (p<0.05) decreased the amounts of diffused
glucose in dialysate within 120 min. After 10 minutes of incubation the
glucose content of the dialysate was reduced invariably by all the fibers as compared to the control (34.59 ± 1.48) but the reduction was highest
in kodo millet AIS (9.04 ± 0.61) and lowest (32.73 ± 1.28) in sorghum
IDF similarly after 30 minutes of incubation the glucose content of the
dialysate was also reduced by all the fibers studied as compared to the
control (79.97 ± 1.34) but the reduction was highest (19.45 ± 1.05) in
kodo millet AIS and lowest (73.08 ± 1.80) in ragi AIS. After 60 minutes
of incubation the reduction in the glucose content of the dialysate was
highest (24.05 ± 1.17) in kodo millet AIS and lowest (100.85 ± 0.99) in
ragi AIS as compared to the control (110.13 ± 1.54). After incubation
for 120 minutes the reduction in the glucose content of the dialysate
was highest (44.25 ± 1.19) in kodo millet WIS and lowest (135.05 ±
1.36) in ragi AIS as compared to the control (144.77 ± 1.07).In case
of kodo millet, proso millet, finger millet and sorghum the glucose
content in dialysate was significantly higher (p<0.05) in IDF than AIS
and WIS. In barnyard millet WIS more glucose (26.89 ± 0.72) was
shown in the dialysate at 10 min than IDF and AIS but at 30, 60 and
120 min glucose diffusion was highest in IDF. In finger millet at 10 min,
glucose concentration was higher in IDF (31.19 ± 1.52) but at 30, 60
and 120 min, it was higher in AIS (73.08 ± 1.80, 100.85 ± 0.99, 135.05 ±
1.36 respectively) than IDF and WIS.

Table 5: Effects of various insoluble fibers on glucose diffusion.

Figure 4: Effects of various insoluble fibers on glucose diffusion.

Effect of insoluble fibers on GDRI (Glucose dialysis retardation
index): The retardation in glucose diffusion by fibers was expressed by
values of GDRI in% (Table 5, Figure 5). GDRI is a useful in vitro index to predict the effect of fiber in the delay in glucose absorption inside
the gastrointestinal tract. Glucose diffusion retardation index (GDRI)
of different fibers after 10 minutes interval varied from 3.92 ± 7.26 in
sorghum IDF to 71.61 ± 3.12 in sorghum WIS and that after 30 minutes
of incubation ranged from 8.34 ± 4.58 in wheat AIS to 75.69 ± 1.17 in
kodo millet AIS. GDRI of different fibers after 60 minutes of incubation
varied from 8.40 ± 0.49 in ragi AIS to 78.12 ± 1.17 in kodo millet AIS
whereas, that after 120 minutes of incubation ranged from 6.72 ± 0.57
in ragi AIS to 69.44 ± 0.71 in kodo millet WIS.

Effect of insoluble fibers on starch digestibility: The effects of
various insoluble fibers on starch digestibility were demonstrated by
changes in the glucose content in dialysate as a function of time when
starch, fiber and diastase were dialysed against distilled water (Table 6, Figure 6). After 10 min of incubation the comparable glucose content
in dialysate with fiber addition indicated that there was significant
difference in starch digestibility in first 10 min in all fibers from six
samples. Kodo millet IDF retarded the starch digestibility to the
greatest extent after 10 min of incubation (glucose in dialysate being
1.84 ± 0.28 micromole) and the wheat IDF retarded the least (glucose
in dialysate being 25.99 ± 0.94 micromole) as compared to the control
value of 31.43 ± 1.08 micromoles of glucose in dialysate. Similarly after
30 min of incubation Kodo millet IDF retarded the starch digestibility
to the greatest extent (glucose in dialysate being 9.43 ± 0.66 micromole) and the porso millet WIS retarded the least (glucose in dialysate being
39.25 ± 1.45 micromole) as compared to the control value of 60.14
± 1.34 micromoles of glucose in dialysate. Kodo millet IDF again
retarded the starch digestibility to the greatest extent after 60 min of
incubation (glucose in dialysate being 18.88 ± 0.98 micromole) and
the sorghum WIS did not retard it at all (glucose in dialysate being
96.57 ± 0.96 micromole) as compared to the control value of 96.17 ±
0.61 micromoles of glucose in dialysate. Further incubation for 120
min porso millet IDF retarded it to the highest extent (glucose in
dialysate being 38.95 ± 1.69 micromole) and the sorghum WIS almost
did not have any effect on it at all (glucose in dialysate being 160.84
± 2.29 micromole) as compared to the control value of 160.84 ± 2.29
micromoles of glucose in dialysate. As the incubation time increases
the results showed that glucose content in the dialysate increased with
subsequent increases of time. The three types of insoluble fibers in all
six samples were found to retard starch digestibility effectively along the
enzymatic digestion process. When compared with control, the glucose
content in the dialysate with all insoluble fibers was less than that of
control excepting the sorghum WIS after 60 min of incubation. In case
of kodo millet, proso millet, and barnyard millet glucose content in
dialysate was higher in WIS than in IDF and AIS but in wheat it was
highest in IDF at 10 and 30 min highest in WIS at 60 and 120 min
whereas, in case of finger millet glucose content in dialysate was highest
in AIS at 10, 30 and 60 min but highest in WIS at 120min.

Figure 6: Effects of various insoluble fibers on starch digestibility.

Effects of various insoluble fibers on alpha-amylase activity: In (Table 7,8, Figure 7,8) the effects of various insoluble fibers on alphaamylase
activity are presented in terms of glucose production rate and
residual amylase activity (%). The reduction in glucose production
rate was lower than that of control in all the samples studied. Glucose
production rate was higher in AIS than IDF and WIS of all the samples.
Among the six samples finger millet have highest glucose production
rate in IDF, AIS and WIS (8.61 ± 1.00, 19.67 ± 1.27 and 18.69 ± 1.56
respectively) and Sorghum IDF showed the lowest value (4.55 ± 0.86).
The reduction in glucose production rate could also be presented in
another way by means of decrease in residual amylase activity (%)
which is the percentage of glucose production rate by addition of fiber
over control. In this case also the AIS of all six samples have higher
residual amylase activity as compared to their corresponding IDF and
WIS, finger millet being the highest (97.67 ± 8.76) and sorghum being
the lowest (53.89 ± 5.99). Sorghum IDF reduced the α-amylase activity
to the lowest extent (21.7 ± 3.32).

Table 7:Effects of various insoluble fibers on starch digestibility.

Table 8: Effect of various insoluble fibers on alpha-amylase activity.

Degenerative diseases such as diabetes mellitus become prevalent
in the population due to more sedentary lifestyle in name of
modernization and westernization. Dietary fiber plays an integral role
in the management of diabetes mellitus. High dietary fiber content
of millets and less prevalence of metabolic disorders in tribal people
regularly consuming it prompted us to undertake a comparative study
on hypoglycemic and anti-oxidative efficacy of insoluble fiber rich
fractions of different cereals and millets commonly grown in tribal
regions of Odessa.

Proximate analysis

Proximate composition analysis was performed for both whole
grains (Table 1) and bran samples (Table 2). As dried and stored
grains were procured so there was slight variation in moisture and dry
matter content in all the grain samples studied (Table 1). The ether
extractives varied from 4.35 ± 0.164% in barnyard millet to 1.003 ±
0.057% in proso millet and high crude fiber content was observed in all
the grains studied (Table 1). The crude protein content of grains ranged
from 10.39 ± 0.248% in barnyard millet and 10.25 ± 0.530% in wheat
to 5.48 ± 0.449% in kodo millet. Ash content also varied from 3.59
± 0.246% in kodo millet and 3.59 ± 0.234% in proso millet to 1.29 ±
0.085% in great millet whereas; the NFE% was almost similar in all the
cases. These types of variations in the composition might be due to the
difference in genetic makeup and environment in different grains and
our results were in accordance to [2,29]. It was clear from the results
that the crude fiber% and the ash content were higher in bran sample
than the whole grain in all six samples studied (Table 2). Hadimani
and Malleshi [3] reported similar findings in different millets. Thus the
bran samples were used for extraction of fiber and in vitro evaluation
of hypoglycemic effect of insoluble fibers whereas, for evaluation of
antioxidant, antibacterial antifungal property and the polyphenol
content whole grain powder was used with methanol as solvent.

Hypoglycemic effect of insoluble fibers from millets in vitro

In vitro hypoglycemic effect of insoluble fibers from different
cereal and millet samples was studied by assessing the effect of various
fibers on glucose adsorption capacity (GAC), glucose diffusion, GDRI
(Glucose dialysis retardation index), starch digestibility and alphaamylase
activity to have a simulation of digestion and absorption of
carbohydrates in the intestine. These experiments were designed to
form the basis of simple methods to measure the potential biological
effects of various insoluble dietary fibers. Discussion of each of these
tests was given separately to have better understanding.

Effect of insoluble fibers on glucose adsorption capacity: (Table 3Figure 1) revealed that all fiber samples at different glucose concentration
(5, 10, 25 and 50 milimol/lit) could bind glucose effectively and amount
of glucose bound to these fibers were concentration dependant and
increased with higher concentration of glucose. There is significant
difference (P<0.05) in the glucose adsorption capacity (GAC) of
different insoluble dietary fibers in all the concentrations of glucose
(Table 3). Ou et al [30] have reported that insoluble fiber derived
from wheat bran could adsorb glucose at different concentrations to
decrease the concentration of glucose available in small intestine. The
finger millet WIS showed highest GAC (0.18 ± 0.01 mM/g) at normal
(5 mM/l) concentration whereas, at a higher glucose concentration of
50 mM/l wheat IDF exhibited highest GAC (1.65 ± 0.02 mM/g). At low
concentration of glucose WIS in all cases showed higher adsorption
of glucose but interestingly it was reversed at higher concentration of
glucose and IDF in all grain samples showed high GAC. Adsorption of
glucose by insoluble fibers might be attributed to the increased water
holding capacity of the fibers. When the glucose concentration reduced
to 5 mmol/litre, the GAC found to be very low which indicated that
the insoluble fiber might help to retain glucose to small extent in the
intestinal lumen even at a low glucose concentration. Our findings
were in binding with the findings of [31]. Further validation of these effects in vivo is required to confirm the hypoglycemic ability of these
fibers.

Effect of insoluble fibers on diffusion of glucose: Diffusion of
glucose from the dialysis membrane was affected by dietary fibers
(Table 4, Figure 2). Diffused glucose for different insoluble fibers was
less affected at 10 and 30 minutes but at 60 and 120 minutes it was
much affected and showed higher retardation in glucose diffusion in
all the cases as compared to control. By hypothesis the effect of dietary
fiber on diffusion was mainly due to their viscosity. The diffusion rate
would decrease as time increased because of the gradual increase in
viscosity of the medium. The diffusion rate of glucose was decreased
by insoluble fibers even if they contribute less to viscosity [30]. This
phenomenon can be explained by adsorption of dietary fiber for glucose.
Some authors indicated that the retardation in glucose diffusion and
absorption due to fibers was affected by viscosity of intestinal content
[32]. The retardation effect of insoluble fibers which contribute little to
viscosity of solution might be probably attributed to their adsorption
capacity. At beginning of dialysis glucose diffusion might be affected
by adsorption of glucose on fiber and viscosity so diffusion rate was
slow (though glucose concentration is higher inside the dialysis bag)
with progress of time diffusion of glucose affected by only viscosity of
fiber. The retardation of glucose diffusion might be due to the physical obstacle presented by fiber particles and entrapment of glucose within
the network of fibers [33,34]. Thus we can conclude that insoluble
fibers have effects delaying glucose diffusion and subsequently
decrease glucose absorption in gastrointestinal tract and millets have
sufficient capacity to reduce glucose diffusion and adsorption inside
the gastrointestinal tract.

Effect of insoluble fibers on glucose dialysis retardation index
(GDRI): (Table 5, Figure 3) showed the variation in GDRI with the
addition of different insoluble fibers. GDRI is a useful in vitro index
to predict the effect of fibers on the delay in the gastro intestinal tract
[34]. After 10 minutes interval, the GDRI of WIS and AIS of all the
samples was significantly (P<0.05) higher than that of IDF. As the time
increased to 30, 60 and 120 min, the trend in the values of GDRI was similar to that of 10 minutes (Table 5). All these results revealed that all
the insoluble fibers could effectively hinder the glucose from diffusing
out of the dialysis membrane and might be efficient in retarding the
glucose absorption. The dialysis experiments mimic events occurring in
the small intestine. Movements in these systems is not by true diffusion
but is assisted by the convective activity of intestinal contractions in
vivo or by stirring of in vitro models [35]. Our experimental design
with stirring simulates the biological system more closely than an
unstirred system. The glucose dialysis studies mimic events occurring
in the jejunum. In this experiment nutrient absorption was modeled
by in vitro measurements of the solute flow from a dialysis bag.
The retardation of the nutrient flow into the external medium is an
indication of the modulating effect of the fibers on glucose absorption in the jejunum. Ability of these fibres to retard the absorption of glucose
in the gastrointestinal tract is a function of their viscosity [36]. The
modest effects on glucose dialysis retardation index seen with insoluble
fibres are in keeping with their effect on glucose absorption in man.
Based on these results, it was conceived that these insoluble fibers could
effectively adsorb glucose, delay the glucose diffusion and subsequently
postpone the glucose absorption in the gastro-intestinal tract.

Effect of insoluble fibers on starch digestibility: The effects of
various insoluble fibers on starch digestibility were presented in (Table
6, Figure 4) IDF of all the grains excepting wheat retarded the starch
digestibility significantly (P<0.05), even at 10 minutes of incubation
there was 70 to >90% retardation of starch digestibility. Similar was
the case with increasing time to 30, 60 and 120 minutes (Table 6).
According to view of [37] dietary fibers can be adsorbed to starch and
thus hinder hydrolysis of starch by alpha-amylase.

The various millet fiber fractions modified enzymatic starch
digestion similarly. Compared to control initial rate of starch digestion
was not modified by the presence of AIS and WIS fibers but a decrease
in final rate and total starch degradation was noted. This decrease
may be attributed to a direct effect of fiber on amylase activity due
to adsorption of enzyme on the fibers or a decrease in activity due
to viscosity or pH modification of medium. Presence of fiber might
have also influenced the accessibility of the enzyme to its substrate.
In the present study, the apparent reduction in the glucose contents
in the dialysate by the presence of insoluble fibers indicated that both
the starch degradation and glucose diffusion could be delayed by the
insoluble fibers of the millets, even though glucose is actually absorbed
into the small intestine through an active process in the human body.

In most of the millets taken, the IDF has more effect on decreasing
starch digestibility. The apparent reduction in glucose content in the dialysate by presence of insoluble fibers (Table 6) indicated that both
the starch degradation and glucose diffusion could be delayed by the
insoluble fibers even through glucose is actually absorbed into the
small intestine through an active process in the human body.

Effect of insoluble fibers on alpha-amylase activity: Effect of
various insoluble fibers from different millet species showed that the IDF
insoluble fibers could exhibit significant (P<0.05) effect in decreasing the
alpha-amylase activity (Table 7, Figure 5 and 6) and starch digestibility
(Table 6, Figure 4) This effect of insoluble fibers of millets might be
due to several possible factors such as fiber concentration, presence of
inhibitor on fibers, capsulation of starch and enzyme by fibers, reduced
accessibility of the starch and direct adsorption of enzyme on fibers
leading to the decrease in amylase activity [31]. The variation in the
inhibitory activity to α-amylase among the different insoluble fibers
suggested that the inhibition depended on the kind of fiber.

In this in vitro study the abilities of insoluble fibers from millet to
adsorb glucose, slowed down glucose diffusion and starch digestibility
and decrease the activity of alpha-amylase suggested that they
might have hypoglycemic effects in delaying release of glucose from
starch, reducing rate of glucose adsorption and hence controlling
concentration of post prandial serum glucose [33].

The potential hypoglycemic effects of these fibers suggested that
they could be incorporated as low-calories bulk ingredient in high fiber
foods to lower post prandial serum glucose level and reduce calories
levels. Further detailed studies are needed to investigate whether the
insoluble fibers from the millets and cereals studied are competent
inhibitors of α-amylase or simply act as a barrier between the amylase
and starch.

• All these grains are having nutritional composition either
comparable or better than most of the commonly used grains
so could be used as staple food.

• All the three types of insoluble fibers from the six millet and
cereal samples taken have potent hypoglycemic effect. Thus
they can be included in diet having low carbohydrate and
high fiber content, which will be beneficial in management of
diabetes both in human being and pet animals.

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